Site-specific glycosylation at Asn-292 in ovalbumin is essential to efficient secretion in yeast

Chicken ovalbumin (OVA) exists as mono-N-glycosylated form with a carbohydrate chain on Asn-292 in egg white, despite the possession of two potential N-glycosylation sites. To investigate the roles of N-glycosylation of OVA, we constructed a series of N-glycosylation mutants deleted N-glycosylation site and compared the secretion level of the mutants in Pichia pastoris. N292Q and N292/311Q mutants resulted in greater lowering of the secretion level as compared with wild-type, whereas N311Q mutant was secreted in approximately equal amounts to wild-type. However, secretion of wild-type and N311Q mutant was inhibited completely by tunicamycin treatment. All the N-glycosylation mutants have been expressed in the cells, as well as wild-type. Circular dichroism and fluorescence spectra of secreted N311Q mutant were almost identical to those of wild-type, while those of N292Q and N292/311Q mutants were different from wild-type; and, N292Q and N292/311Q mutants showed considerably lower denaturation temperature than wild-type. The results indicate that N-glycosylation at Asn-292 of OVA is required for the folding and secretion.     

Body Mass Index and Mortality among Korean Elderly in Rural Communities: Kangwha Cohort Study

BackgroundThe relationship between body mass index (BMI) and mortality may differ by ethnicity, but its exact nature remains unclear among Koreans. The study aim was to prospectively examine the association between BMI and mortality in Korean.Methods6166 residents (2636 men; 3530 women) of rural communities (Kangwha County, Republic of Korea) aged 55 and above were followed up for deaths from 1985–2008. The multivariable-adjusted hazard ratios were calculated using the Cox proportional hazards model.ResultsDuring the 23.8 years of follow-up (an average of 12.5 years in men and 15.7 years in women), 2174 men and 2372 women died. Men with BMI of 21.0–27.4 and women with BMI of 20.0–27.4 had a minimal risk for all-cause mortality. A lower BMI as well as a higher BMI increased the hazard ratio of death. For example, multivariable-adjusted hazard ratios associated with BMI below 16, and with BMI of 27.5 and above, were 2.4 (95% CI = 1.6–3.5) and 1.5 (95% CI = 1.1–1.9) respectively, in men, compared to those with BMI of 23.0–24.9. This reverse J-curve association was maintained among never smokers, and among people with no known chronic diseases. Higher BMI increased vascular mortality, while lower BMI increased deaths from vascular diseases, cancers, and, especially, respiratory diseases. Except for cancers, these associations were generally weaker in women than in men.ConclusionsA reverse J-curve association between BMI and all-cause mortality may exist. BMI of 21–27.4 (rather than the range suggested by WHO of 18.5–23 for Asians) may be considered a normal range with acceptable risk in Koreans aged 55 and above, and may be used as cut points for interventions. More concern should be given to people with BMI above and below a BMI range with acceptable risk. Further studies are needed to determine ethnicity-specific associations.     

Deamino-hydroxy-phoslactomycin B, a biosynthetic precursor of phoslactomycin, induces myeloid differentiation in HL-60 cells

During the screening for novel differentiation inducers, we found that a culture broth of Streptomyces sp. HK-803 induced myeloid differentiation of HL-60 cells. The active substance was identified as deamino-hydroxy-phoslactomycin B (HPLM) by mass spectrometry, and synthesized HPLM also induced the differentiation of HL-60 cells. HPLM showed greater inhibition of protein phosphatase 2A (PP2A) activity than phoslactomycin B (PLMB); however, PLMB and okadaic acid did not induce differentiation. Moreover, treatment with ATRA and 1α, 25(OH)₂D₃ induced retinoic acid receptor-β and 1α, 25(OH)₂ D₃ 24-hydroxylase, respectively, whereas HPLM did not, suggesting that HPLM is a novel differentiation inducer.     

Fly pollination in Ceropegia (Apocynaceae: Asclepiadoideae): biogeographic and phylogenetic perspectives

Background and Aims

Ceropegia (Apocynaceae subfamily Asclepiadoideae) is a large, Old World genus of >180 species, all of which possess distinctive flask-shaped flowers that temporarily trap pollinators. The taxonomic diversity of pollinators, biogeographic and phylogenetic patterns of pollinator exploitation, and the level of specificity of interactions were assessed in order to begin to understand the role of pollinators in promoting diversification within the genus.

Methods

Flower visitor and pollinator data for approx. 60 Ceropegia taxa were analysed with reference to the main centres of diversity of the genus and to a cpDNA–nrDNA molecular phylogeny of the genus.

Key Results

Ceropegia spp. interact with flower-visiting Diptera from at least 26 genera in 20 families, of which 11 genera and 11 families are pollinators. Size range of flies was 0·5–4·0 mm and approx. 94 % were females. Ceropegia from particular regions do not use specific fly genera or families, though Arabian Peninsula species are pollinated by a wider range of Diptera families than those in other regions. The basal-most clade interacts with the highest diversity of Diptera families and genera, largely due to one hyper-generalist taxon, C. aristolochioides subsp. deflersiana. Species in the more-derived clades interact with a smaller diversity of Diptera. Approximately 60 % of taxa are so far recorded as interacting with only a single genus of pollinators, the remaining 40 % being less conservative in their interactions. Ceropegia spp. can therefore be ecological specialists or generalists.

Conclusions

The genus Ceropegia has largely radiated without evolutionary shifts in pollinator functional specialization, maintaining its interactions with small Diptera. Intriguing biogeographic and phylogenetic patterns may reflect processes of regional dispersal, diversification and subsequent specialization onto a narrower range of pollinators, though some of the findings may be caused by inconsistent sampling. Comparisons are made with other plant genera in the Aristolochiaceae and Araceae that have evolved flask-shaped flowers that trap female flies seeking oviposition sites.Key words: Apocynaceae, Asclepiadoideae, Brachystelma, Ceropegia, Diptera, flower evolution, generalization, mutualism, pollination, Riocreuxia, specialization, Stapeliinae     

Saponin inhibits hepatitis C virus propagation by up-regulating suppressor of cytokine signaling 2

Saponins are a group of naturally occurring plant glycosides which possess a wide range of pharmacological properties, including anti-tumorigenic and antiviral activities. To investigate whether saponin has anti-hepatitis C virus (HCV) activity, we examined the effect of saponin on HCV replication. HCV replication was efficiently inhibited at a concentration of 10 μg/ml of saponin in cell culture grown HCV (HCVcc)-infected cells. Inhibitory effect of saponin on HCV replication was verified by quantitative real-time PCR, reporter assay, and immunoblot analysis. In addition, saponin potentiated IFN-α-induced anti-HCV activity. Moreover, saponin exerted antiviral activity even in IFN-α resistant mutant HCVcc-infected cells. To investigate how cellular genes were regulated by saponin, we performed microarray analysis using HCVcc-infected cells. We demonstrated that suppressor of cytokine signaling 2 (SOCS2) protein level was distinctively increased by saponin, which in turn resulted in inhibition of HCV replication. We further showed that silencing of SOCS2 resurrected HCV replication and overexpression of SOCS2 suppressed HCV replication. These data imply that saponin inhibits HCV replication via SOCS2 signaling pathway. These findings suggest that saponin may be a potent therapeutic agent for HCV patients.     

Intragastric ascorbic but not uric acid is depleted in relation with the increased pH in patients with atrophic body gastritis and H. pylori gastritis

Background. Helicobacter pylori gastritis induces reversible lowering of Ascorbic Acid (AA) intragastric concentrations. No studies have been aimed at determining the gastric juice AA concentration of atrophic body gastritis (ABG) patients. Uric Acid (UA), is another potent hydro‐soluble scavenger of ROS and its possible modification in the gastric juice of patients with H. pylori gastritis have never been investigated. This study was aimed at investigating the levels of AA and UA in the plasma and gastric juice of ABG patients, compared with H. pylori positive patients without corporal atrophy, and with healthy individuals. Materials and Methods. Thirteen ABG patients (Group 1): 32 Chronic non‐atrophic H. pylori gastritis patients (Group 2); and 13 healthy stomach controls (Group 3) attending gastroscopy with gastric biopsies (antrum = 3, corpus = 3) had plasma and intragastric levels of AA and UA measured. Results. Intragastric AA concentration was significantly lower in group 1 (median 0.21 µg/ml, range 0.1–24) compared both with groups 2 (median 5.5 µg/ml, range 0.1–33.2) (p = 0.043) and 3 (median 14.9 µg/ml, range 0.34–44.8) (p = 0.0028). Intragastric UA was not different between the three groups. Intragastric AA concentration resulted negatively correlated with the intragastric pH (Spearman r = ?0.47, p = 0.0003). In patients with gastritis (groups 1 and 2) there was a significant negative correlation between the sum of the Sydney Score variables in the body mucosa, and AA in the gastric juice (Spearman r = ?0.55; p = 0.0001). Conclusion. The study shows that intragastric pH is the key factor for the depletion of gastric juice AA observed in patients with corporal atrophy and to a lower extent with nonatrophic H. pylori gastritis.     

Statins lower plasma and lymphocyte ubiquinol/ubiquinone without affecting other antioxidants and PUFA

It has been shown that treating hypercholesterolemic patients (HPC) with statins leads to a decrease, at least in plasma, not only in cholesterol, but also in important non-sterol compounds such as ubiquinone (CoQ10), and possibly dolichols, that derive from the same biosynthetic pathway. Plasma CoQ10 decrease might result in impaired antioxidant protection, therefore leading to oxidative stress. In the present paper we investigated the levels in plasma, lymphocytes and erythrocytes, of ubiquinol and ubiquinone, other enzymatic and non-enzymatic lipophilic and hydrophilic antioxidants, polyunsaturated fatty acids of phosfolipids and cholesterol ester fractions, as well as unsaturated lipid and protein oxidation in 42 hypercholesterolemic patients treated for 3 months. The patients were treated with different doses of 3 different statins, i.e. atorvastatin 10 mg (n = 10) and 20 mg (n = 7), simvastatin, 10 mg (n = 5) and 20 mg (n = 10), and pravastatin, 20 mg (n = 5) and 40 mg (n = 5). Simvastatin, atorvastatin and pravastatin produced a dose dependent plasma depletion of total cholesterol (t-CH), LDL-C, CoQ10H2, and CoQ10, without affecting the CoQ10H2/CoQ10 ratio. The other lipophilic antioxidants (d-RRR-alpha-tocopherol-vit E-, gamma-tocopherol, vit A, lycopene, and beta-carotene), hydrophilic antioxidants (vit C and uric acid), as well as, TBA-RS and protein carbonyls were also unaffected. Similarly the erythrocyte concentrations of GSH and PUFA, and the activities of enzymatic antioxidants (Cu,Zn-SOD, GPx, and CAT) were not significantly different from those of the patients before therapy. In lymphocytes the reduction concerned CoQ10H2, CoQ10, and vit E; other parameters were not investigated. The observed decline of the levels of CoQ10H2 and CoQ10 in plasma and of CoQ10H2, CoQ10 and vit E in lymphocytes following a 3 month statin therapy might lead to a reduced antioxidant capacity of LDL and lymphocytes, and probably of tissues such as liver, that have an elevated HMG-CoA reductase enzymatic activity. However, this reduction did not appear to induce a significant oxidative stress in blood, since the levels of the other antioxidants, the pattern of PUFA as well as the oxidative damage to PUFA and proteins resulted unchanged. The concomitant administration of ubiquinone with statins, leading to its increase in plasma, lymphocytes and liver may cooperate in counteracting the adverse effects of statins, as already pointed out by various authors on the basis of human and animal studies.     

Modal gating of human CaV2.1 (P/Q-type) calcium channels: II. the b mode and reversible uncoupling of inactivation

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445-461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.